vector to knockout E. coli genes

R.D. Haigh rxh at leicester.ac.uk
Mon Jul 13 10:24:38 EST 1998

>Doug Stemke

I'm looking for recommendations for an E. coli vector for knocking out
an E.coli gene (I have the entire gene already sequenced, I just need
to make a null mutant). I believe I am looking for something that I
can confirm knockouts by altering temperature of the culture and
arresting plasmid replication and then screen the mutants using
selective antibiotic sensitivities. It would also be nice if it had a
nice polyclonal region as well, but not entirely necessary as I can
PCR in restriction sites.

Recently everyone in Gram negatives seems to have shifted over to 
using SacB dependant systems for making null mutants. You can look up 
the references but the system basically consists of a two step 
procedure; you clone your mutant gene into a suicide plasmid which contains a positive
selection marker eg Amp and a negative selection SacB. You transfer 
into the target strain and select for Amp - this will select for 
homologous recombination of the plasmid into the chromosome at the 
site of your gene. The resultant merodiploid can then be "resolved" 
by selection on Sucrose media - sacB is lethal in E.coli and most 
other Gram negatives when grown on sucrose - resolution can have one 
of two results i. the wildtype gene is reconstructed and the mutant is lost in the 
plasmid or ii. the wildtype is lost in the plasmid and the mutant is 
constructed in the chromosome.  I hope this makes sense if not have a 
look at the papers.
We have (like everyone else it seems) constructed our own vector and 
are using it regularily for knockouts with reasonable degrees of 
success - there are problems with sacb selection some genes just will 
not play usually due to problems with growth on the selective media.

These are useful refs but as you will see on a quick search using 
sacB everyone is using it now.

Allelic exchange in Escherichia coli using the Bacillus subtilis sacB
gene and a temperature-sensitive pSC101 replicon. Blomfield IC, Vaughn
V, Rest RF, Eisenstein BI Mol Microbiol 1991 Jun 5:6 1447-57

Construction of an eae deletion mutant of enteropathogenic Escherichia
coli by using a positive-selection suicide vector. Donnenberg MS,
Kaper JB Infect Immun 1991 Dec 59:12 4310-7

If you are interested I can send info on our plasmid or I have the 
plasmid Jim Kaper used for the  above paper.


    Richard Haigh                      
    Department of Microbiology and Immunology,                        
    University of Leicester,
    University Road,        
    Leicester. LE1 9HN UK
    Phone: 0116 2523017 
    FAX   : 0116 2525030 
    Email : rxh at nospam.le.ac.uk
    NB. remove nospam to Email  

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